1. Field of the Invention
The present invention relates to a method of controlling the structure stability of collagen fibers produced from solutions or suspensions of collagen which have been treated with a chemical reagent for the deactivation of infectious agents.
2. Description of the Background Art
There are public health issues pertaining to the use of animal and human tissues in medical devices which are implanted into human beings, to the use of such tissues in pharmaceuticals, and to a lesser degree, to use in cosmetics. Of particular concern are slow acting viruses present in such tissues, which slow viruses are particularly difficult to deactivate.
U.S. Pat. No. 4,511,653 to Play et al., issued Apr. 16, 1985, describes a process for the industrial preparation of human collagenous material from human placental tissue. This process includes subjecting the placental tissue to an alkaline treatment with a 0.5M solution of NaOH, a 0.5M solution of potassium hydroxide (KOH), or a saturated lime water solution at a temperature of less than or equal to 10.degree. C., for purposes of inactivation of viruses.
In June of 1986, "Concise Communications", The Journal of Infectious Diseases, Vol. 153, No. 6, there is a description of the inactivation of slow acting viruses, such as scrapie virus and CJD virus present in 20% brain homogenates, using various concentrations of sodium hydroxide (NaOH) for one hour at room temperature.
In September of 1991, "Recommendations for Minimizing the Risk of Infection by Agents Causing Zoonoses and Other Animal Infections in Manufacture of Medicinal Products", Federal Journal of Official Publications (BAnz., Germany), No. 164, p 6120, there is the description of the treatment of medical materials with a solution of 1N (1M) NaOH for one hour at 20.degree. C. for the purpose of inactivation of infectious agents. This treatment was recommended particularly for application to bovine spongiform encephalopathy (BSE) and materials of bovine origin.
In 1992, "Public Health Issues Related to Animal and Human Spongiform Encephalopathies": Memorandum from a WHO Meeting, Bulletin of the World Health Organization, 70(2): pp 183-190, a discussion is presented regarding BSE, a member of the group of transmissible spongiform encephalopathies (THE) whose prototype is scrapie. Treatment of medicinal products derived from bovine tissues with NaOH, preferably 1M, for 1 hour at 20.degree. C. is recommended as a manufacturing process for removal or reduction of BSE infectivity.
Collagen Corporation, assignee of the present application, produces a variety of products having bovine collagen as a principal component. The source of Collagen's bovine collagen is a closed herd which is controlled to reduce the potential of contamination by a source of THE (or other infectious agent). It is questionable whether THE is present within the U.S.; however, due to the problem in foreign countries and the possibility of contamination of U.S. bovine supplies, it is desirable to have a method of treating animal tissues used in the preparation of implantable medical devices, medicinal products, and cosmetics. The sodium hydroxide (NaOH) treatment of such tissues has been demonstrated to be particularly effective in the reduction of infectious agents in general. With this in mind, the evaluation of a processes for NaOH treatment of collagen solutions and suspensions used to prepare collagen-based products was carried out.
Collagen may be obtained in commercially useful amounts from the connective tissues of a variety of domesticated animals, such as cattle and swine, for example. The native collagen is most conveniently obtained from tendons or skin and is freed from extraneous matter such as lipids, saccharides and non collagen protein, so as to leave the collagen protein free or substantially free of other connective tissue materials. Native collagen fibers are composed of regularly arranged subunit structures referred to as collagen molecules. Each collagen molecule is about 3000 .ANG. long and 15 .ANG. in diameter. This long rigid rod-like structure consists of three polypeptide chains wound together in a triple helical configuration. Typically two of the constituent chains are identical in composition and the third is different. A characteristic distribution of amino acid residues along the length of any given polypeptide strand, wherein repeating triplets contain glycine at every third position, favors the formation of a helical configuration. The individual collagen units form fibrils which associate to form fibers.
The nonhelical terminal portions of the native collagen molecule, the telopeptides, exhibit a preferred coil conformation extending from the amino and carboxy ends of the molecule. These telopeptides appear to serve a number of functions in the formation of the native collagen fiber. The telopeptides serve as the primary sites for crosslinking intramolecularly (between the three constituent polypeptide chains in the native collagen molecule) and intermolecularly (between two or more native collagen molecules). In addition, the telopeptides facilitate the arrangement of the individual collagen molecules in a pattern which provides for the regular structure of native fibrous collagen. However, the telopeptide portions of native (heterogenic) collagen are believed to be the major sites of its immunogenicity. Therefore, in order to minimize the immunogenicity of heterogenic collagen, it is desirable that the telopeptides be removed. This leaves the collagen fibers in a less stable, more fragile condition, and in need of protection when protection when exposed to processing conditions, which can disturb the arrangement (association) of collagen molecules within collagen fibers.
Typically collagen is obtained from bovine hides. The initial stage is to clean the hide physically so as to remove some of the noncollagen materials, such as hair, fat, carbohydrates, mucopolysaccharides and the like. See, for example, U.S. Pat. Nos. 2,934,446 and 3,121,049, as well as Chvapil et al., "Medical and Surgical Applications of Collagen", Connective Tissue Research 4 (1973).
To enhance the ease of purification and facilitate the enzymatic removal of the telopeptides, the collagenous material is subjected to various mechanical treatments, such as dissection, grinding, high speed shearing, milling and the like. Depending upon the particular treatment, the collagen may be wet or dry, frozen or cooled, with grinding and high speed shearing preferably being wet processes, and milling being a dry process.
Coarsely divided connective tissues are swollen in aqueous acidic solutions under nondenaturing conditions. Further dispersion is achieved through extensive wet grinding, to facilitate enzyme access to the native collagen. Preferably dilute acid solutions at low temperatures are employed to minimize denaturation. Suitable acids are acetic, malonic or lactic acids, or other lyotropic carboxylic acids having pK values from about 2 to about 5 at 25.degree. C. Concentrations of acid in the dispersion medium range from about 0.01M to 1.0M, and temperatures may vary from about 4.degree. C. to about 25.degree. C. The dispersion which is obtained by treatment with acid is a viscous dispersion containing native collagen microaggregates and a small amount of native collagen in solution.
The viscous product is subjected to enzymatic treatment to remove the telopeptides and to produce soluble atelopeptide collagen. Various proteolytic enzymes may be employed which preferentially attack the telopeptides, while leaving the major portion of the molecule intact. Illustrative enzymes include pepsin, trypsin and pronase, for example. See U.S. Pat. Nos. 3,131,130 and 3,530,037.
The preferred enzyme is pepsin, which is used in combination with an acidic solution, generally at a pH of about 2 to 4. The concentration of the enzyme varies from about 0.001 to about 10 weight percent based on the weight of collagen present. The collagen concentration generally varies from 0.5 g/l. to about 10 g/l. Preferably, the acidity is provided by a carboxylic acid in a concentration of about 0.01M to about 1M. If necessary, the pH can be adjusted by the addition of a mineral acid, e.g. hydrochloric acid. The enzymatic treatment is generally carried out over temperatures ranging from about 0.degree. C. to about 30.degree. C. over a time period ranging between two days and two weeks, with progress monitored periodically until substantially complete solubilization of the collagen is achieved.
The resulting solution is treated to separate the soluble atelopeptide collagen from insoluble collagen, enzymes, residual amino acids, and the telopeptide units which have been separated from the collagen molecules. Primarily, the treatment involves separations, precipitations and dialysis against various solutions of different ionic strength. Moderate temperatures are employed, normally from about 4.degree. C. to about 30.degree. C., and salt solutions of various ionic strength or concentration are employed, generally from about 0.01M to 3.5M, depending upon the particular salt. Ionic strengths are usually about 0.01 to 3.5.
Conveniently, the solution is treated with an alkaline material, e.g., sodium hydroxide, to raise the pH of the solution to at least about seven, to inactivate the enzyme. After inactivating the enzyme, non-solubilized contaminants which have been precipitated during the inactivation treatment are filtered off to yield a filtrate which contains collagen in solution.
The collagen in solution is passed through a bed of celite and subsequently processed via ultrafiltration to provide a purified, clear solution containing about 3 mg/ml of atelopeptide collagen. This concentrated solution of collagen is relatively free of higher aggregates, and is referred to as concentrated submicron filtrate (CSF).
From a virus deactivation/inactivation point of view, it is preferable to treat a solution of collagen with sodium hydroxide for virus inactivation prior to the formation of the fibrous micropolymers, since the collagen molecules and any beginning fibrils contained in the solution are dissociated to permit maximum availability of any infectious agents which may reside in or be trapped within fiber structures. The collagen triple helix is too tightly wound (1.5 nm diameter) for a virus to reside within the collagen molecule. Therefore, such virus would be present either in the solution or absorbed onto the surface of a collagen molecule. In the soluble environment, where collagen fibers are dissociated into collagen molecules, there is no mass transfer barrier, which requires the sodium hydroxide to diffuse through solids (assembled fibers) to reach the infectious agents on the surface of collagen molecules.
During the evaluation of NaOH treatment of collagen solutions and suspensions, it was discovered that the structure of the collagen fibers formed was affected by the 1.5 NaOH treatment. The fibrillar collagen produced from solutions and suspensions of NaOH treated collagen exhibited significantly different polymeric characteristics.
To be able to use the NaOH treatment of collagen solutions for deactivation of infectious agents, it was necessary to find a method to compensate for the effects of the treatment, enabling production of the desired fibrillar collagen product. The fibrous collagen product had to be biocompatible and sufficiently stable that it was relatively insensitive to contact with chemical additives and to processing necessary to achieve final product formulation.